Duck pneumovirus and corresponding vaccines

ABSTRACT

An immunogenic preparation or vaccine against avian pneumovirosis, comprising an antigen of the avian pneumovirus strain C990427, in a vehicle or excipient which is acceptable from the veterinary point of view and, optionally, an adjuvant.

[0001] The present invention relates to a novel strain of pneumovirus,to live attenuated vaccines, to inactivated vaccines, to methods forvaccinating birds, and in particular ducks, against a pneumovirus, andto methods for diagnosing infection with a pneumovirus.

[0002] Infection of chickens and turkeys with avian pneumoviruses (orAPVs) is known. In these species, pneumoviruses cause seriousrespiratory syndromes affecting the upper respiratory tract, loss ofweight, or even death of these animals. This pneumovirosis is also namedturkey rhinotracheitis (TRT) and swollen head syndrome (SHS) in chicken,the latter also being characterized by the appearance of subcutaneousedemas on the head of the animal. Avian pneumoviruses can also causedrops in egg production in layers. The economic consequences of thesediseases are considerable.

[0003] It is the same virus, named turkey rhinotracheitis virus (orTRTV), which causes these two diseases.

[0004] The Pneumovirus genus belongs to the family Paramyxoviridae, thisfamily also comprising the genera Paramyxovirus, Morbillivirus andRubulavirus. Pneumoviruses are nonsegmented single-stranded RNA virusesof negative polarity. They are enveloped and their capsid is helical.Unlike other Paramyxoviridae (in particular the Newcastle disease virusor NDV), Pneumoviruses possess neither hemagglutinin nor neuraminidase(Alexander D. J., Vet. Microbiol., 1990, 23, 103-114).

[0005] Avian pneumoviruses have been isolated from chicken or turkeys inFrance, Great Britain, Italy, Spain, Hungary, Germany, the Netherlands,Greece, Brazil, Mexico, Morocco, South Africa, Taiwan and Israel. Avirus antigenically and genetically different from previously knownviruses was also isolated in turkeys in the United States at thebeginning of 1997. This virus is often named Colorado virus (Seal B. S.,Virus Research, 1998, 58, 45-52; Ali et al., Avian Disease, 1999, 43,600-603).

[0006] The various isolates of these avian pneumoviruses show a geneticand antigenic diversity which may allow the strains to be classified inat least two subgroups named A and B (Juhasz et al., J. Gen. Virol.,1994, 75, 2873-2880). That article cites some avian pneumovirus strains,in particular the strains APV 2119 (of subgroup B, originating fromItaly), 657/4 (subgroup B, originating from Hungary), 872S (of subgroupB, originating from Spain) and CVL 14/1 (of subgroup A, originating fromthe United Kingdom).

[0007] The Colorado APV strain belongs to neither subgroup A norsubgroup B (Seal B. S., Virus Research, 1998, 58, 45-52).

[0008] Vaccination against avian pneumoviruses has allowed prevention ofdisease in turkeys and chickens. Live and inactivated vaccines areavailable. Vaccine strains may be from subgroup A or from subgroup B andhave been isolated from turkeys or chickens. Live vaccines areattenuated on cell cultures. Live vaccines are generally used in theyoung in meat birds and reproducers. Inactivated vaccines are adjuventedand are indicated in booster vaccination in reproducer birds or in egglayers.

[0009] To date, only chickens and turkeys have been described as naturalhosts for avian pneumoviruses. Experimental infection experiments withan avian pneumovirus of turkey origin (strain CVL 14/1, from subgroup A,described in Wyeth et al., Vet. Rec., 1986, 119, 139) have shownsensitivity to the disease and an antibody response in turkeys, chickensand pheasants. Chickens and turkeys show symptoms of upper respiratorytract infections (nasal drip, sneezing, etc.) and pheasants showsymptoms of slight conjunctivitis.

[0010] In these experiments, the pigeon, the goose and the mallard duck(Anas platyrhynchos) appeared to be refractory to the turkey pneumovirus(Cough et al., Vet. Rec. 1988, 123, 58-59).

[0011] The applicant has revealed a novel avian pneumovirus for whichthe preferred host is duck.

[0012] This virus was isolated in France from organ samples originatingfrom a flock of female reproducer mallard ducks of the Peking type (Anasplatyrhynchos). The animals of this flock exhibited symptoms of egg dropand of mortality.

[0013] Ground material from organs was inoculated onto cultures ofspecific pathogen Free chick embryo cells (CECs). At the 10th passage onCECs, a pneumovirus was identified by immunofluorescence with a serumdirected against APV strain Colorado. The immunofluorescence testscarried out with sera specific for other duck or chicken virusesremained negative (duck parvovirus, duck plague virus, duck reovirus,avian group I adenovirus, infectious bronchitis virus, Newcastle diseasevirus, egg drop syndrome-76 adenovirus, avian influenza virus,paramyxovirus type 3, infectious bursal disease virus, avianencephalomyelitis virus). An immunofluorescence test carried out withantisera specific for various avian pneumovirus strains of subgroup Aand of subgroup B also proved to be negative. Moreover, the virusisolated by the applicant is not a hemagglutinating virus.

[0014] A paramyxovirus has been visualized by electron microscopy incultures of CECs and of Vero cells infected with the virus.

[0015] These elements indicate that a novel avian pneumovirus whichinfects ducks has been isolated. This virus is antigenically differentfrom the other turkey and chicken avian pneumoviruses of subgroups A andB previously known, and is distinguished from the APV strain Colorado byits host specificity.

[0016] The virus was isolated and cultured on a CEC culture, and wasthen adapted on a Vero cell culture. Passaging on Vero cell cultures maybe used to attenuate the virus.

[0017] A subject of the present invention is a culture of this novelvirus (name C990427) as deposited, on Nov. 25, 1999, with the CollectionNationale de Cultures de Micro-organismes (or CNCM) [National Collectionof Micro-organism Cultures] of the Pasteur Institute, Paris, France,under the accession number I-2353.

[0018] A subject of the present invention is also the immunogenicpreparations and the vaccines against avian pneumovirosis, comprising atleast one antigen or immunogen of the APV strain C990427 in a vehicle orexcipient which is acceptable from a veterinary point of view and,optionally, in the presence of an adjuvant.

[0019] The notion of immunogenic preparation herein covers anypreparation capable, once administered to birds, of inducing an immuneresponse directed against the avian pathogen under consideration. Theterm “vaccine” is intended to mean a preparation capable of inducingeffective protection. The vaccines according to the invention make itpossible to prevent and/or treat the infection.

[0020] Like the other avian pneumoviruses already known, this virus maybe used to immunize or vaccinate birds, in particular gallinaceans, e.g.turkeys, hens and chickens, and more particularly ducks, especiallymallard ducks (Anas platyrhynchos) and Muscovy ducks (Cairina moschata),against infection caused by avian pneumoviruses, and in particularagainst infection caused by duck pneumoviruses.

[0021] According to a first mode, a subject of the invention is a liveattenuated immunogenic preparation or a live attenuated vaccine.

[0022] The novel virus may be attenuated by the usual methods known tothose skilled in the art, in particular by 10 to 150 passages on primarycell culture, for example on CEC culture, or on cell lines, for exampleon Vero cells, or on embryonated eggs. The number of passages ismultiplied until there is a lack of significant symptoms in animals.

[0023] To produce the vaccinal virus, the attenuated virus may then bemultiplied on primary cells, for example CECs, or cells from a line, forexample on Vero cells, or on embryonated eggs. The immunogenicpreparation or the vaccine incorporating a suitable amount of virusmultiplied in this way may be frozen or lyophilized with a stabilizer,in particular SPGA (EP-B1-0008255). Each vaccinal dose may contain from10² to 10⁶ 50% cell culture infectious doses (CCID50) per dose, andpreferably from 10² to 10⁴.

[0024] The attenuated immunogenic preparation or the attenuated vaccineaccording to the invention may also be mixed with other viral orbacteria valences so as to constitute multivalent immunogenicpreparations or multivalent vaccines. The viral or bacterial valencesare selected from avian pathogens characteristic of the avian species tobe vaccinated. They are in particular selected from the group comprisingNewcastle disease virus (NDV), infectious bronchitis virus—(IBV),infectious bursal disease virus (IBDV), duck parvovirus and the otheravian pneumoviruses, including TRTV. These other valences may inparticular be conventional attenuated valences or recombined valences.

[0025] The attenuated immunogenic preparations and attenuated vaccinesmay be administered to birds individually by inoculation either in ovo,in particular between approximately 4 and 1 day(s) before hatching, andpreferably 3 days before hatching, or via eyedrops, by dipping the beak,or by intramuscular or subcutaneous injection.

[0026] The attenuated immunogenic preparations and attenuated vaccinesmay be administered collectively via drinking water or nebulization.

[0027] The immunogenic preparation or vaccine may be administered afterhaving been taken up in an adjuvented diluent, for example a diluentadjuvented with an aqueous adjuvant such as alumina hydroxide.

[0028] According to a second mode, a subject of the invention is aninactivated immunogenic preparation or an inactivated vaccine.

[0029] The virus may be multiplied on primary cells, for example CECs,or cells from a line, for example from Vero cells, or on embryonatedeggs. Before or after inactivation, the virus thus produced may beclarified, in particular by filtration or by centrifugation, and/or maybe concentrated, in particular by ultra-filtration or precipitation,and/or may be purified, in particular by selected precipitation or bychromatography. The inactivation may be carried out by the usual methodsknown to those skilled in the art, in particular by chemical treatment(eg. formaldehyde, β-propiolactone, ethyleneimine, binary ethyleneimine(BEI)) and/or heat treatment. The inactivated virus may then be mixedwith an adjuvant, preferably an aqueous adjuvant, for example based onalumina hydroxide, or may be formulated in the form of an emulsion, inparticular an water-in-oil emulsion, composed of mineral oil ormetabolizable oil and one or more nonionic surfactants. By way ofexample, the water-in-oil emulsion comprises polysorbate 80 (e.g. Tween®80) in the aqueous phase, and mineral oil (e.g. Drakeol® 6-VR) andsorbitan monooleate (e.g. Span® 80) in the oily phase (Stone et al.,Avian Dis., 1978, 22, 666-674).

[0030] Each vaccinal dose may contain the equivalent of 10³ to 10⁸CCID50 per dose, and preferably from 10⁵ to 10⁷, before inactivation.The amount of antigen per dose may also be determined by other methodsusing antigen/anti-body reactions, for example by the ELISA method.

[0031] The inactivated antigen may also be mixed with other viral orbacterial valences so as to constitute multi-valent immunogenicpreparations or multivalent vaccines. The viral or bacterial valencesare selected from the avian pathogens characteristic of the avianspecies to be vaccinated. They are in particular selected from the groupcomprising Newcastle disease virus (NDV), infectious bronchitis virus(IBV), infectious bursal disease virus (IBDV), egg drop syndrome virus,duck parvovirus, paramyxovirus type 3 and the other avian pneumoviruses,including TRTV. These valences may in particular be conventionalinactivated valences, recombined valences or subunit-based valences.

[0032] The inactivated immunogenic preparations and inactivated vaccinesare preferably administered to birds by intramuscular or subcutaneousinjection. The inactivated antigen may also consist of or comprisefractionated or subunit antigens.

[0033] A subject of the present invention is also compositionscomprising the novel pneumovirus C990427 in live attenuated form or ininactivated form, optionally a vehicle or excipient which is acceptablefrom a veterinary point of view and, optionally, an adjuvant.

[0034] The contents of C990427 and the choice of adjuvants are asdescribed above.

[0035] A subject of the present invention is also compositionscomprising fractionated or subunit antigens of C990427, optionally avehicle or excipient which is acceptable from a veterinary point of viewand, optionally, an adjuvant.

[0036] A subject of the invention is also methods for immunizing orvaccinating birds, in particular ducks and gallinaceans such as hens,chickens and turkeys, against diseases due to avian pneumoviruses, andin particular against diseases due to duck pneumoviruses, methods inwhich an immunogenic preparation or a vaccine according to the inventionis administered to the animal.

[0037] The programs for immunizing or vaccinating meat ducks may inparticular comprise one or two administration(s) to animalsapproximately 1 to approximately 3 weeks old, with an attenuatedvaccine, intramuscularly or subcutaneously, preferably with an adjuvant,in particular an aqueous adjuvant such as alumina hydroxide, orocularly, preferably without adjuvant. A booster administration may begiven between 2 and 4 weeks after the first administration. Preferably,when the ducks are intended for reproduction, they are alsoadministered, around the age of 10 weeks, a new dose of live vaccineand, before the egg laying period, an inactivated vaccine adjuvented inthe form of an emulsion, intramuscularly or subcutaneously.

[0038] For the other avian meat species, in particular turkeys andchickens, a live attenuated, preferably nonadjuvented, vaccine ispreferably administered once or twice to young animals, in the drinkingwater or by nebulization. The first administration may be performed onanimals approximately 14 days old. A booster administration may be givenbetween 2 and 4 weeks after the first administration.

[0039] Preferably, when the other avian species, in particular turkeysand hens, are intended for reproduction or for egg laying, they are alsoadministered, before the egg laying period, an inactivated vaccine whichis adjuvented, preferably in the form of an emulsion, intramuscularly orsubcutaneously.

[0040] Those skilled in the art have the competence necessary to defineprecisely the number of injections and the doses of each vaccine to beused for each vaccination protocol.

[0041] The dose volumes may in particular be between 0.1 and 0.8 ml,preferably of the order of 0.3 to 0.5 ml.

[0042] A subject of the present invention is also the use of thepneumovirus C990427, for preparing an immunogenic preparation or avaccine intended to be administered to a bird, in particular a duck,according to the invention, said preparation or vaccine also comprisinga vehicle or excipient which is acceptable from a veterinary point ofview and, optionally, an adjuvant.

[0043] A subject of the present invention is also the use of an antigenor immunogen of the pneumovirus C990427, for preparing an immunogenicpreparation or a vaccine intended to be administered to a bird, inparticular a duck, according to the invention, said preparation orvaccine also comprising a vehicle or excipient which is acceptable froma veterinary point of view and, optionally, an adjuvant.

[0044] The invention is also directed toward such a use in the contextof preparing a combined immunogenic preparation or a combined vaccine,or in the context of a combined vaccination program.

[0045] The invention also relates to the reagents used for diagnosinginfection with the avian pneumovirus C990427.

[0046] This virus may be used, firstly, as a crude or purified antigenor antigen in subunit form and, secondly, in crude, purified or subunitform for immunizing animals for the purpose of producing polyclonal andmonoclonal antibodies. The antigens and the antibodies can be used inthe diagnostic method according to the invention and for developingdiagnostic kits, in particular of the ELISA type.

[0047] The invention will now be described in greater detail usingembodiments taken by way of nonlimiting examples.

EXAMPLES Example 1

[0048] Isolation of the Virus

[0049] Organs (firstly, liver, heart, oviduct, trachea and spleen mixedtogether and, secondly, intestines) were removed from female reproducermallard ducks of the Peking type (Anas platyrhynchos) exhibitingsymptoms of egg drop and of mortality. The two batches of organs wereground separately in phosphate buffer (PBS) supplemented with anantibiotic ({fraction (1/10000)} of gentamycin), frozen, thawed and thenclarified by centrifugation. The centrifugation supernatant was dilutedagain in order to obtain a final dilution of the organs of approximately{fraction (1/10)}th (W/V) for the mixture of liver, heart, oviduct,trachea and spleen, and of approximately {fraction (1/50)}th (W/V) forthe intestines.

[0050] The ground materials were filtered (0.22 μm).

[0051] A culture of primary SPF (specific pathogen free) chick embryocells (CEC1) was prepared by inoculation of 3×10⁶ cells per 25-cm² flaskin F10-199 medium, pH 6.9 to 7.1, supplemented with 5% of fetal calfserum (FCS). The cells were incubated at 38° C. with 5% of CO₂.

[0052] The composition of the F10-199 medium is as follows: 60 ml of F10(10 times concentrated), 40 ml of 199 Hanks (10 times concentrated), 40ml of TPB, 20 ml of 5.6% bicarbonate, 1 ml of vitamins and water qs for1000 ml.

[0053] After 24 h of culture, the medium was discarded and a mixture ofequal parts of the two ground materials was inoculated onto the CEC1cells, in a proportion of 0.8 ml per 25-cm² flask. After 30 min ofcontact at 38° C., the cells were rinsed with culture medium. Culturemedium supplemented with 1% of FCS was then added and the cultures werereincubated as previously (1st passage).

[0054] After three days of culture, the cells were treated with pronase,and 3×10⁶ cells were reinoculated per 25-cm² flask in F10-199 mediumsupplemented with 3% of FCS, in order to obtain a secondary CEC culture(CEC2). The cultures were reincubated as previously for seven days (2ndpassage) and were then frozen and thawed and the suspension thusobtained was clarified by centrifugation.

[0055] 1 ml of the supernatant was inoculated onto a new layer of CEC1and the culture was continued as previously (3rd passage).

[0056] Further passages, alternately on CEC2 and on CEC1, were thencarried out.

[0057] At the 4th passage, some clusters of rounded and refringent cellswere observed after four days of culture. From the 5th passage, plaquesof rounded and refringent cells and syncitia were observed. From the10th passage, a generalized cytopathic effect (CPE) was observed after 3days of culture.

Example 2

[0058] Adaptation of the Virus on Vero Cells

[0059] A culture of the virus at the 11th passage on CECs, prepared inexample 1, was frozen, thawed and clarified by centrifugation.

[0060] A culture of Vero cells was prepared by inoculation of 10⁶ cellsper 25 cm² flasks in modified eagle medium (MEM), pH 6.9 to 7.1,supplemented with 5% of FCS. The cells were incubated at 38° C. with 5%of CO₂.

[0061] The composition of the MEM medium is as follows: 200 ml of MEMEarle's salt (5 times concentrated), 0.1 ml of 1% biotin, 10 ml of 200mM glutamine, 1.5 ml of phenol red, 10 ml of 10% glucose, 30 ml of 5.6%bicarbonate and water qs for 1000 ml.

[0062] After 24 h of culture, the medium was discarded and the virusoriginating from the 11th passage on CECs was inoculated in a proportionof 1 ml of the centrifugation supernatant described above per 25-cm²flask. These cells were reincubated as previously. After 6 days ofculture, clusters of rounded and refringent cells and syncitia wereobserved and lysis plaques began to appear on the carpet of cells.

[0063] The subsequent passages on Vero cells were carried out byfreezing, thawing and clarifying the cultures, and then inoculating thesupernatant into a new culture of Vero cells in a proportion of 0.5 mlof supernatant per 25-cm² flask. The cells were reincubated aspreviously.

[0064] Each culture was harvested when the CPE became generalized,approximately 3 to 6 days after the beginning of the culture.

[0065] The viral suspension may be stored at a temperature below orequal to −40° C. before it is subsequently used.

Example 3

[0066] Characterization of the Virus by Immunofluorescence

[0067] The virus isolated according to example 1 was identified byimmunofluorescence on cell cultures. Vero cell cultures in 96-wellmicroplates were prepared, in a proportion of 1.8×10⁴ cells in 0.2 ml ofMEM medium, pH 6.9 to 7.1, supplemented with 3% of FCS, per well.

[0068] After 24 h of incubation at 38° C. with 5% of CO₂, the medium wasdiscarded and the virus was inoculated in a proportion of approximately200 CCID50 in 0.2 ml of MEM medium, pH 6.9 to 7.1, supplemented with 1%of FCS. The plates were incubated as previously for 48 to 72 h. Whenlysis plaques began to appear on the carpet of cells, the cultures wererinsed by washing with PBS. A solution of acetone (100 ml of pureacetone+15 ml of distilled water) was added to all the wells in order tofix the cells. The plates were placed at −20° C. for 20 minutes. Theacetone was then discarded and the plates were dried. The plates thusfixed can be stored at −20° C.

[0069] Sera monospecific for avian pneumovirus of subgroup A, for avianpneumovirus of subgroup B, for avian pneumovirus strain Colorado, forduck parvovirus, for duck plague virus, for avian reovirus, for aviangroup I adenovirus, for infectious bronchitis virus, for Newcastledisease virus, for egg drop syndrome-76 virus, for avian influenzavirus, for paramyxovirus type 3; for infectious bursal disease virus andfor avian encephalomyelitis virus were diluted separately to {fraction(1/20)} in physiological saline. Each serum was brought into contact,separately, with the infected Vero cells, in a proportion of 100 μl ofdiluted serum per well.

[0070] The plates were placed in an incubator at 38° C. for 60 minutes,then the sera were removed and the plates were rinsed 3 times withphysiological saline and 3 times with demineralized water.

[0071] A commercial anti-chicken fluorescent conjugate was diluted tothe concentration recommended by the supplier (RACH FITC conjugate,Nordic), and then added in a proportion of 50 μl per well containing achicken virus. A commercial anti-duck fluorescent conjugate was dilutedto the concentration recommended by the supplier (RADU EITC conjugate,Nordic) and then added in a proportion of 50 μl per well containing aduck virus.

[0072] The plates were incubated for 30 minutes at 38° C., then theconjugate was removed and the plates were rinsed 3 times withphysiological saline and 3 times with demineralized water.

[0073] The plates thus obtained were observed under fluorescencemicroscope. Intense intracytoplasmic fluorescence was observed with theserum specific for avian pneumovirus strain Colorado, whereas noreaction was observed with the other sera.

Example 4

[0074] Electron Microscopy

[0075] CEC (example 1) or Vero cell (example 2) cultures were inoculatedwith the virus. When the CPE became visible, the carpet cells wereprefixed with glutaraldehyde, underwent osmic acid fixation, and weredehydrated with ethyl alcohol.

[0076] The areas of interest exhibiting the beginnings of CPE were thenembedded in epoxy resin and ultrathin sections of the samples were cutand contrasted.

[0077] The observations were made using a transmission electronmicroscope. Penetration of the viruses by fusion of the viral envelopewith the cell membrane, intracytoplasmic accumulation of nucleoproteincompounds and budding of viral particles at the level of the cytoplasmicmembrane were observed. Polymorphous mature enveloped viral particleswere observed in the extracellular space. Some particles are rounded andhave a diameter equal to a minimum of 130 nm. Other particles arefilamentous and are greater than 1000 nm in size. Bulges sometimesexhibiting spikes are also observed. These images are characteristics ofthe structure and of the morphogenesis of Paramyxoviridae.

Example 5

[0078] Production of the Virus on Vero Cells

[0079] After thawing, the virus originating from the stock described inexample 2 can be cultured in roller bottles.

[0080] Vero cells were prepared in MEM medium, pH 6.9 to 7.1,supplemented with 5% of FCS. The virus was mixed with the cells with amultiplicity of infection (MOI) of 1 to 0.01, and the mixture wasdistributed into roller bottles in a proportion of 120×10⁶ cells perroller bottle. The bottles were incubated at 38° C. When the CPE causedby the virus was optimal (more than 50% of the carpet of cellsdestroyed), the viral suspension was harvested after vigorous shaking ofthe bottles.

[0081] The harvest may be mixed at 50% with a stabilizer, for example abuffered solution. The viral suspension may be stored at a temperatureof below or equal to −40° C. before it is subsequently used.

Example 6

[0082] Titering the Virus on Vero Cells

[0083] The titering was performed in 96-well microplates in MEM medium,pH 6.9 to 7.1, supplemented with 3% of FCS. Approximately 10-folddilutions of virus were mixed with a suspension of Vero cells in themicroplates, in a proportion of 0.1 ml of virus dilution per 1.8×10⁴cells in 0.15 ml per well. After 6 to 9 days of incubation at 38° C.with 5% of CO₂, the wells exhibiting a generalized CPE were counted andthe titer was calculated as 50% cell culture infectious doses (CCID50)by the Karber method.

Example 7

[0084] Preparation of a Live Vaccine

[0085] After thawing of the virus obtained in example 5, the amount ofvirus was adjusted to the desired titer.

[0086] An SPGA stabilizer (sucrose, phosphate, glutamate and albumin,EP-B1-0008255) was added to the viral suspension, which was distributedinto bottles and lyophilized. The vaccinal virus was titered as inexample 6. The titer of the vaccine was adjusted to 10², 10³ and 10⁴CCID50 per dose.

[0087] Before it is administered, the vaccinal virus may be taken up inan adjuvented diluent containing alumina hydroxide. The adjuvant iscomposed of an isotonic phosphate buffer containing 2.1 grams of aluminahydroxide per liter. The lyophilized vaccine was taken up in the diluentsuch that 0.5 ml of diluent contains one dose of vaccinal virus.

Example 8

[0088] Preparation of an Inactivated Vaccine

[0089] The virus was cultured according to the method described inexample 5. The viral suspension was clarified by centrifugation. Thevirus was inactivated with formaldehyde.

[0090] A 40% formaldehyde solution was added so as to obtain a finalconcentration of formaldehyde of 0.1% in the viral suspension. Themixture was maintained at 37° C. for 12 h with moderate stirring, andthen cooled to 5° C.

[0091] The viral suspension obtained may be concentrated 10 to 50-foldby ultrafiltration. The inactivated viral suspension, which may or maynot be concentrated, may be stored at a temperature of 5° C. before itis used.

[0092] A water-in-oil emulsion is prepared by slowly adding the aqueousphase to the oily phase, over 15 min, with moderate agitation using aturbomixer. Homogenization was carried out for 10 min at 15 to 25 m/sec,and then the emulsion thus formed was cooled to 5° C. and stored at thistemperature.

[0093] The aqueous phase contains the inactivated virus and 3.2% v/v ofpolysorbate 80 (Tween® 80).

[0094] The oily phase contains 10% v/v of sorbitan monooleate (Span® 80)and 90% v/v of mineral oil (Drakeol® 6-VR).

[0095] The aqueous phase to oily phase ratio is 1:4.

[0096] The viral suspensions for the inactivated vaccine comprise10^(6.2), 10^(7.2) and 10^(8.2) CCID50 per ml.

Example 9

[0097] Vaccination Method and Program

[0098] In young ducks, the adjuvented live vaccine prepared as describedin example 7 is administered subcutaneously in a proportion of one doseof 0.5 ml in the first week of life and a booster of a dose of 0.5 mlbetween 2 and 4 weeks after the first administration.

[0099] In reproducer ducks, the above program is used, and then abooster with the live vaccine is given around the 10th week of age. Abooster with a dose of 0.3 ml of oily inactivated vaccine (example 8) isthen given before each period of egglaying.

Example 10

[0100] Method of Diagnosis

[0101] The diagnosis of infection is carried out by viral isolationaccording to the method as described in example 1 or by serology using aseroneutralization method.

[0102] The seroneutralization is performed on Vero cell cultures in96-well microplates. The sera to be tested are serially dilutedfour-fold in MEM medium, pH 6.9 to 7.1, supplemented with 1% of FCS,directly in the microtitration plates. One well per serum and perdilution is used, at a volume of 0.1 ml of diluted serum per well. Thevirus, used at a concentration of 200 CCID50 in 0.025 ml, is added toeach well. A contact time of 30 minutes at 38° C. is used. The VEROcells are added to each well in a proportion of 1.8×10⁴ cells in 0.15 mlof MEM medium, pH 6.9 to 7.1, supplemented with 1% of FCS. A negativeserum, a control positive serum and also cell controls are set up foreach reaction. Reading is carried out after 9 days of incubation at 38°C. with 5% of CO₂, under plastic film. The approximate percentageinhibition of the CPE (0%, 50%, 100%) is assessed. The last dilutionwhich causes complete inhibition of the CPE expresses the 100% titer ofthe serum tested. The last dilution which causes 50% inhibition of theCPE expresses the 50% titer of the serum tested.

[0103] It should be clearly understood that the invention defined by theattached claims is not limited to the particular embodiments indicatedin the above description, but encompasses the variants which departneither from the context nor the spirit of the present invention.

1. A culture of avian pneumovirus named C990427 and deposited with theCNCM under the accession number I-2353.
 2. An immunogenic preparation orvaccine against avian pneumovirosis, comprising the avian pneumovirusC990427, a sample of which is deposited with the CNCM under theaccession number I-2353, in a vehicle or excipient which is acceptablefrom the veterinary point of view and, optionally, an adjuvant.
 3. Theimmunogenic preparation or vaccine as claimed in claim 2, characterizedin that it comprises live attenuated virus as antigen.
 4. Theimmunogenic preparation or vaccine as claimed in claim 3, characterizedin that it comprises an aqueous adjuvent, preferably alumina hydroxide.5. The immunogenic preparation or vaccine as claimed in claim 3,characterized in that it comprises from 10² to 10⁶ CCID50 per dose,preferably from 10² to 10⁴.
 6. The immunogenic preparation or vaccine asclaimed in claim 3, characterized in that it also comprises at least oneantigen originating from at least one other avian pathogenic agent,preferably selected from the group consisting of Newcastle diseasevirus, infectious bronchitis virus, infectious bursal disease virus,duck parvovirus and avian pneumoviruses.
 7. The immunogenic preparationor vaccine as claimed in claim 2, characterized in that it comprisesinactivated virus as antigen.
 8. The immunogenic preparation or vaccineas claimed in claim 7, characterized in that it comprises an aqueousadjuvant, preferably alumina hydroxide, or is formulated in the form ofan emulsion, preferably a water-in-oil emulsion.
 9. The immunogenicpreparation or vaccine as claimed in claim 8, characterized in that itis formulated in the form of a water-in-oil emulsion containingpolysorbate 80 in the aqueous phase, and mineral oil and sorbitanmonooleate in the oily phase.
 10. The immunogenic preparation or vaccineas claimed in claim 7, characterized in that it comprises from 10³ to10⁸ CCID50 per dose, preferably from 10⁵ to 10⁷, before inactivation.11. The immunogenic preparation or vaccine as claimed in claim 7,characterized in that it also comprises at least one antigen originatingfrom another avian pathogenic agent, preferably selected from Newcastledisease virus, infectious bronchitis virus, infectious bursal diseasevirus, egg drop syndrome adenovirus, duck parvovirus, paramyxovirus typeIII and avian pneumoviruses.
 12. A method for immunizing or vaccinatingbirds, in particular ducks and gallinaceans such as hens, chickens andturkeys, against diseases due to avian pneumoviruses, in which animmunogenic preparation or a vaccine as claimed in claim 2 isadministered to the bird.
 13. The method as claimed in claim 12,intended for vaccinating meat birds, in which unattenuated vaccine isadministered once or twice to animals approximately 1 to 3 weeks old,intramuscularly or subcutaneously or ocularly and, eventually a boosteris administered between 2 and 4 weeks after the first administration.14. The method as claimed in claim 13, in which the administration iscarried out intramuscularly or subcutaneously and the attenuated vaccinecomprises an aqueous adjuvant such as alumina hydroxide.
 15. The methodas claimed in claim 13, in which the administration is carried outocularly.
 16. The method as claimed in claim 12, intended for immunizingor vaccinating ducks for reproduction, which method comprises one or twoadministrations to ducks approximately 1 to 3 weeks old, with anattenuated vaccine, intramuscularly or subcutaneously or ocularly,optionally a booster administration between 2 and 4 weeks, and then anadministration at 10 weeks after the first administration, while, beforethe egglaying period, they are administered an adjuvented inactivatedvaccine intramuscularly or subcutaneoulsy.
 17. The method as claimed inclaim 16, characterized in that the inactivated vaccine is adjuvented inthe form of an emulsion.
 18. The method as claimed in claim 18, in whichthe attenuated vaccine is administered intramuscularly or subcutaneoulsyand is adjuvented with an aqueous adjuvant such as alumina hydroxide.19. A method for immunizing or vaccinating meat turkeys and chickens, inwhich a live attenuated vaccine, as claimed in claim 3, is administeredonce or twice in the drinking water or by nebulization.
 20. The methodas claimed in claim 19, in which the first administration is performedon animals approximately 14 days old and a booster administration isgiven between 2 and 4 weeks after the first administration.
 21. A methodfor immunizing or vaccinating turkeys and chickens intended forreproduction or for egglaying, in which a live attenuated vaccine, asclaimed in claim 3, is administered once or twice to young animals, inthe drinking water or by nebulization, and then an adjuventedinactivated vaccine, as claimed in claim 7 is administered, before theegglaying period, intramuscularly or subcutaneously.
 22. The method asclaimed in claim 21, in which the first administration of liveattenuated vaccine is performed on animals approximately 14 days old andthe second booster administration is given 2 to 4 weeks after the firstadministration.
 23. The method as claimed in claim 21, in which theinactivated vaccine is adjuvented in the form of an emulsion.
 24. Theuse of the pnuemovirus C990427 deposited with the CNCM under theaccession number I-2353, for producing an immunogenic preparation or avaccine intended to be administered to birds, in particular poultry,such as ducks and gallinaceans.
 25. The use of an antigen or immunogenof the pneumovirus C990427 deposited with the CNCM under the accessionnumber I-2353, for producing an immunogenic preparation or a vaccineintended to be administered to birds, in particular poultry, such asducks and gallinaceans.
 26. The use as claimed in claim 24, intended foradministration in ducks.
 27. An immunogenic preparation or vaccineagainst avian pneumoviruses, comprising an antigen specific for theavian pneumovirus C990427, a sample of which is deposited with the CNCMunder the accession number I-2353, in a vehicle or excipient which isaccepatable from a veterinary point of view and, optionally, anadjuvant.
 28. The use as claimed in claim 25, intended foradministration in ducks.